It has been reported that several prokaryotic plasmids have a post-segregation killing (PSK) function to kill hosts from which the plasmids have been dropped out in order to maintain the plasmids in the hosts. Such plasmids have toxin-antitoxin genes. An antitoxin binds to a toxin in a cell to inactivate the toxin. The antitoxin is labile to degradation by proteases. Degradation of the antitoxin by proteases results in activation of the toxin which is stable (Non-patent Document 1). Such toxin-antitoxin genes also exist on chromosomes of most prokaryotes. They respond to various stresses and have functions in programmed cell death. Although the functions of the toxins have not been fully proven, it has been suggested that CcdB and ParE may control replication targeting DNA gyrase, and RelE and Doc may control transcription (Non-patent Documents 1 and 2).
At least five toxins RelE, ChpAK (MazF), ChpBK, YoeB and YafQ exist in Escherichia coli (Non-patent Document 2). Christensen et al. have reported that RelE is an endoribonuclease that recognizes a specific codon of three nucleotides in a ribosome-dependent manner to cleave mRNA (Non-patent Documents 3 and 4). Furthermore, Christensen et al. have reported that ChpAK, ChpBK and YoeB are also endoribonucleases that cleave mRNA in a manner dependent on ribosome and codon (Non-patent Documents 5 and 6).
Inouye et al. have demonstrated that MazF (ChpAK) is an endoribonuclease that recognizes specific nucleotides ACA in a ribosome-independent manner to cleave mRNA (Non-patent Documents 7 and 8). Munoz-Gomez et al. have reported that the cleavage of RNA with mazF is specific for NAC (Non-patent Document 9). Inouye et al. have demonstrated that PemK in a plasmid R100 is an endoribonuclease that recognizes specific nucleotides UAH (H is C, A or U) to cleaves mRNA (Patent Document 1, Non-patent Document 10). As described above, it has been suggested that toxins of the RelE or PemK family may be endoribonucleases that cleave mRNA in a nucleotide-specific manner. In particular, toxins of the PemK family may be endoribonucleases that recognize specific nucleotides in a ribosome-independent manner to cleave mRNA. Many toxins of the PemK family exist in prokaryotes and comparison of their sequences has been studied extensively (Non-patent Documents 1 and 11).
Anantharaman et al. have phylogenetically classified toxins by conducting gene neighborhood analyses on the basis of genetic information about toxins and genetic information about organisms for which genomic analyses have been completed, and predicted toxin-like proteins from proteins of unknown functions (Non-patent Document 12). Furthermore, it has been suggested through the analyses that not only RelE and PemK but also proteins of the Doc family and proteins having PIN domains may have ribonuclease activities. Five toxins of the PemK family have been found in Nitrosomonas europaea (Non-patent Document 13).
As to enzymes that cleave nucleic acids in a sequence-specific manner, many restriction enzymes which cleave double-stranded DNA have been found and widely utilized in the field of genetic engineering. As to enzymes that cleave single-stranded RNA in a sequence-specific manner, ribonuclease T1 which specifically cleaves at a G nucleotide has been found and utilized for genetic engineering (Non-patent Document 14). The number of enzymes that recognize plural nucleotides in single-stranded RNA and specifically cleave it is still small. Development of such endoribonucleases has been desired in the field of genetic engineering. If an endoribonuclease that specifically recognizes and cleaves a sequence of three nucleotides (like MazF) or more than three nucleotides is found, it is considered that the endoribonuclease would become a useful enzyme in the field of genetic engineering.
Patent Document 1: WO 2004/113498
Non-patent Document 1: J. Bacteriol., 182:561-572 (2000)
Non-patent Document 2: Science, 301:1496-1499 (2003)
Non-patent Document 3: Molecular Microbiol., 48:1389-1400 (2003)
Non-patent Document 4: Cell, 122:131-140 (2003)
Non-patent Document 5: J. Mol. Biol., 332:809-819 (2003)
Non-patent Document 6: Molecular Microbiol., 51:1705-1717 (2004)
Non-patent Document 7: Molecular Cell, 12:913-920 (2003)
Non-patent Document 8: J. Biol, Chem., 280:3143-3150 (2005)
Non-patent Document 9: FEBS Letters, 567:316-320 (2004)
Non-patent Document 10: J. Biol. Chem., 279:20678-20684 (2004)
Non-patent Document 11: J. Mol. Microbial. Biotechnol., 1:295-302 (1999)
Non-patent Document 12: Genome Biology, 4:R81 (2003)
Non-patent Document 13: Nucleic Acids Research, 33:966-976 (2005)
Non-patent Document 14: Methods in Enzymology, 341:28-41 (2001)